Fig 2: Restoration of COL4A2 in HCC cell reversed the effects of HES4 knockdown under hypoxia. (A) CCK-8 assays were performed to detect the cell viability of indicated cells. (B) EDU positive rate in indicated cells was detected. (C) Wound healing assays were performed to detect the cell migration of indicated cells. **P < 0.01

Fig 3: HES4 was key hypoxia-biomarker for HCC. (A) DEG analysis for the HCC tissues with high and low hypoxia score. (B) The 34 DEGs were also identified as key genes related to hypoxia through WGCNA. (C) Among the 34 DEGs, HES4 was also identified to up-regulate in HCC tissues compared to adjacent tissues. (D) HCC patients with high HES4 had shorter overall survival rate. (E) The HES4 mRNA levels were elevated in HCC tissues compared to adjacent non-tumor tissues from our research cohort. (F) HES4 mRNA had high diagnostic value for distinguish HCC tissues. (G) The protein levels of HES4 were up-regulated in HCC tissues in comparison to adjacent tissues. (H) The mRNA levels of HES4 were elevated in HCC tissues provided from patients in stage III-IV compared with those provided from patients in stage I-II. (I) The protein levels of HES4 were elevated in HCC tissues provided from patients with high AFP levels compared with those provided from patients with low AFP levels. (J) qRT-PCR experiments indicated that hypoxia increased the mRNA levels of HES4. (K, L) Western blotting experiments indicated that hypoxia increased the protein levels of HES4. **P < 0.01

Fig 4: Knockdown of HES4 suppressed the promotive effects of hypoxia on HCC cell proliferation and mobility. (A) HES4 knockdown cell were constructed. (B) CCK-8 experiments were used to detect the cell viability of NC cell and HCC cell with HES4 knockdown under normoxia and hypoxia. (C) EDU positive rate was detected in NC cell and HCC cell with HES4 knockdown under normoxia and hypoxia. (D) Wound healing assay experiments were used to detect the cell migration of NC cell and HCC cell with HES4 knockdown under normoxia and hypoxia. (E) Transwell assays were used to detect the cell invasion of NC cell and HCC cell with HES4 knockdown under normoxia and hypoxia. **P < 0.01

Fig 5: COL4A2 was co-expressed with HES4 in HCC tissues. (A) DEGs between HCC tissues with high and low HES4 expression were analyzed. (B) Top 50 up-regulated and down-regulated DEGs were exhibited. (C) KEGG terms of up-regulated DEGs were analyzed. (D) COL1A1, COL1A2 and COL4A2 were found to link ECM receptor interaction, focal adhesion, ACE-PAGE signaling, and PI3K-AKT signaling. (E) Co-expression relationship between HES4 for COL1A1, COL1A2 and COL4A2. (F, G) COL4A2 expression was up-regulated in HCC tissues with high HES4. (H) HES4 and COL4A2 protein were co-expressed in HCC tissues. **P < 0.01